A C-13-NMR STUDY OF THE ROLE OF ASN-155 IN STABILIZING THE OXYANION OF A SUBTILISIN TETRAHEDRAL ADDUCT

By removing one of the hydrogen-bond donors in the oxyanion hole of su btilisin BPN, we have been able to determine how it affects the cataly tic efficiency of the enzyme and the pK(a) of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN c ontains five mutations which enhance its stability. Site-directed muta genesis was used to prepare the N155A mutant of this variant. The cata lytic efficiencies of wild-type and variant 8397 are similar, but repl acing Asn-155 with alanine reduces catalytic efficiency approx. 300-fo ld. All three forms of subtilisin were alkylated using benzyl- bonylgl ycylglycyl[2-C-13]phenylalanylchloromethane and examined by IRC-NMR. A single signal due to the C-13-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahed ral adduct formed between the hydroxy group of Ser-221. and the inhibi tor. This signal had chemical shifts in the range 98.3-103.6 p.p.m., d epending on the pH. The titration shift of 4.7-4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pK(a) values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pK(a) increased to 8.09. We explain why such a small increase is observed and we conclude that it is the inte raction between the oxyanion and the imidazolium cation of the active- site histidine that is the main factor responsible for lowering the ox yanion pK(a).